Polynucleotides encoding cellulase enzymes from Piromyces rhizinflata

Abstract

The invention relates to a cellulase enzyme, elgA, isolated from the fungus Piromyces rhizinflata and nucleic acids encoding it.

Description

BACKGROUND OF THE INVENTION

Cellulases are enzymes that can hydrolyze the glycosidic linkages in polysaccharides such as cellulose. These enzymes are used in a number of industrial applications where breaking down biomass is beneficial. For example, cellulases can be used as a supplement in animal feed to decrease the production of fecal waste by increasing the digestibility of the feed. Cellulases can also be used to increase the efficiency of alcoholic fermentations (e.g., in beer brewing) by converting undigestible biomass into fermentable sugars. In addition, the "softening" of blue jeans to produce a "stone-washed" look can be facilitated by treating the jeans with cellulases.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new cellulase isolated from the fungus Piromyces rhizinflata. The gene encoding this cellulase is designated eglA. A portion of an eglA cDNA is described below.

Accordingly, the invention features a substantially pure polypeptide having an amino acid sequence at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to an amino acid sequence representing the catalytic domain of EGLA (SEQ ID NO:4; described below), the polypeptide encoded by eglA. The polypeptide is capable of hydrolyzing a polysaccharide containing a β-1,3' or β-1,4' glycosidic linkage. Such a polysaccharide can be cellulose (e.g., carboxymethyl cellulose), polysaccharides containing both β-1,3' and β-1,4' glycosidic linkage (e.g., barley β-glycan), or lechinan.

The invention also includes an isolated nucleic acid encoding a polypeptide of the invention. For example, the invention includes an isolated nucleic acid having a sequence encoding a polypeptide that hydrolyzes a polysaccharide containing a β1,3' or β1,4' glycosidic linkage, provided that the nucleic acid hybridizes under stringent conditions to SEQ ID NO:1.

In addition, the invention features any vectors or transformed cells which contain a nucleic acid of the invention. Vectors include nucleic acid vectors, such as expression plasmids, or viral vectors. Transformed cells include eukaryotic and prokaryotic cells.

A "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized or modified) DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. An "isolated nucleic acid" refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the 5' non-coding (e.g., promoter) sequences which are immediately contiguous to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. The term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

By "hybridizes under stringent conditions" is meant specific and non-covalent binding to an immobilized reference nucleic acids in the presence of 0.2×SSC (1.75 g/l NaCl, 0.88 g/l Na₃ citrate. 2H₂ O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68°C

The term "substantially pure" as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) by weight pure. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity or conservation to a reference polypeptide or nucleic acid, the percent identity or conservation is determined by the algorithm of Myers and Miller, CABIOS (1989), which is embodied in the ALIGN program (version 2.0), or its equivalent, using a gap length penalty of 12 and a gap penalty of 4 where such parameters are required. All other parameters are set to their default positions. Access to ALIGN is readily available. See, e.g., http://www2.igh.cnrs.fr\/bin/align-guess.cgi on the Internet.

Other features or advantages of the present invention will be apparent from the following detailed description, the drawings, and also from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a cellulase enzyme, nucleic acids encoding it, and vectors and cells containing such nucleic acids. Contemplated within the scope of this invention are recombinant nucleic acids or viruses which allow production of EGLA in a transformed cell or transgenic organism or allow ease of producing specific or non-specific mutations within the EGLA reading frame. These recombinant nucleic acids or viruses may further include any one of a variety of sequences flanking or within the EGLA coding sequences, such as strong constitutive promoters within the EGLA coding sequence, as introns containing cis-elements that allow high level expression, or efficient polyadenylation signals.

Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the isolation of EGLA polypeptides and nucleic acids described below, utilize the present invention to its fullest extent. The following examples are to be construed as merely illustrative of how one skilled in the art can isolate and use EGLA polypeptides and nucleic acids from biological sources, and are not limitative of the remainder of the disclosure in any way. For example, once the sequence of the egla CDNA is known, any egla sequence can be obtained by PCR amplification of mRNA or genomic DNA. Any publications cited in this disclosure are hereby incorporated by reference.

The anaerobic fungus Piromyces rhizinflata, strain 2301, was cultivated anaerobically at 39°C in a modified semi-defined medium as described in Lowe et al., J. Gen. Microbiol. 131:2225-2229, 1985. The mycelia were harvested from the culture media, lyophilized, frozen in liquid nitrogen, and ground into a powder. The powder was homogenized in extraction buffer containing 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl, 2% SDS, and 1% β-mercaptoethanol. An equal volume of a 1:1 mixture of phenol/chloroform was added, and the resulting mixture vortexed for 60 seconds and then centrifuged. The aqueous phase was extracted with the phenol/chloroform again. A one-third volume of 8 M LiCl was then added to the extracted mixture. The mixture was centrifuged sufficiently to pellet the RNA, which was washed with 2 M LiCl, followed by 80% ethanol. The washed RNA was then resuspended in diethyl pyrocarbonate (DEPC)-treated water.

Polyadenylated RNA was isolated from total RNA using a standard oligo-(dT)-cellulose chromatography column. The construction of a cDNA expression library was carried out using a Stratagene kit. The library was screened for cellulase activity by overlaying plaques with 0.7% (w/v) agarose containing 0.2% (w/v) carboxymethyl cellulose (CMC). The plates were incubated at 39°C overnight, then stained with a 0.1% (w/v) aqueous solution of Congo red and destained with 1 M NaCl as described in Teather et al., App. Environ. Microbiol. 43:777-780, 1982. Cellulase-producing plaques were surrounded by a clear halo visible against a red background. The positive clones were excised and purified using standard procedures. One of the clones, designated pPr2301-10, was selected for further study. The mRNA and gene from which the cDNA residing in pPr2301-10 was designated eglA.

The complete sequence of the cDNA insert in plasmid pPr2301-10 was determined using a commercial service (Bio S&T, Lachine, QC, Canada). Translation of one reading frame revealed a 1748 bp open reading frame (ORF), as shown below.

1          GG CAC GAG CTT GAA TGG AAC ATT AAT TTA ATG AAG AAA AGA TTT GTT
GAT CAA GGT 56
1             H   E   L   E   W   N   I   N   L   M   K   K   R   F   V   D
Q   G 18
57     ATT CCA ATG ATT CTT GGT GAA TAT GGT GCT ATG AAC CGT GAC AAT GAA GAA
GAT CGT GCT        116
19     I   P   M   I   L   G   E   Y   G   A   M   N   R   D   N   E   E
D   R   A 38
117    ACT TGG GCT GAA TTC TAC ATG GAA AAG GTT ACT GCT ATG GGA GTT CCA CAA
ATC TGG TGG        176
39     T    W   A   E   F   Y   M   E   K   V   T   A   M   G   V   P   Q
I   W   W 58
177    GAT AAT GGT ATC TTC CAA GGT ACT GGT GAA CGT TTT GGT CTT CTT GAT CGT
AAG AAC TTA        236
59     D   N   G   I   F   Q   G   T   G   E   R   F   G   L   L   D   R
K   N   L 78
237    AAG ATT GTT TAT CCA ACT ATT GTT GCT GCT TTA CAA AAG GGT AGA GGT TTA
GAA GTT AAT        296
79     K   I   V   Y   P   T   I   V   A   A   L   Q   K   G   R   G   L
E   V   N 98
297    GTT GTT CAT GCT GTT GAA AAA AAA CCA GAC GAA CCA ACT AAA ACT ACC AAA
CCA ACT GAA        356
99     V   V   H   A   V   E   K   K   P   D   E   P   T   K   T   T   K
P   T   E 118
357    CCA ACT GAA ACT ACT AGT CCA GAA GAA TCA ACT AAG CCA GAA GAA CCA ACT
GGT AAT ATC        416
119    P    T   E   T   T   S   P   E   E   S   T   K   F   E   E   P   T
G   N   I 138
417    CGT GAT ATT TCA TCA AAG GAA TTG ATT AAG GAA ATG AAT TTC GGT TGG AAT
TTA GGT AAT        476
139    R   D   I   S   S   K   E   L   I   K   E   M   N   F   G   W   N
L   G   N 158
477    ACT ATG GAT GCT CAA TGT ATT GAA TAC TTA AAT TAT GAA AAG GAT CAA ACT
GCT TCA GAA        536
159    T    M   D   A   Q   C   I   E   Y   L   N   Y   E   K   D   Q   T
A   S   E 178
537    ACT TGC TGG GGT AAT CCA AAG ACT ACT GAA GAT ATG TTC AAG GTT TTA ATC
GAC AAC CAA        596
179    T   C   W   G   N   P   K   T   T   E   D   M   F   K   V   L   I
D   N   Q 198
597    TTT AAT GTC TTC CGT ATT CCA ACT ACT TGG TCT GGT CAC TTC GGT GAA GCT
CCA GAT TAT        656
199    F    N    V    F    R    I    P    T    T    W    S    G    H    F
G    E     A   P    D    Y 218
657    AAG ATT GAT GAA AAA TGG TTA AAG AGA GTT CAT GAA GTT GTT GAT TAT CCA
TAC AAG AAC        716
219    K    I    D    E    K    W    L    K    R   V    H    E    V    V
D    Y    P    Y    K    N 238
717    GGA GCA TTT GTT ATC TTA AAT CTT CAT CAT GAA ACC TGG AAT CAT GCC TTC
TCT GAA ACT        776
239    G    A    F    V    I    L    N    L    H    H    E    T    W    N
H    A    F    S    E   T 258
777    CTT GAT ACA GCC AAG GAA ATT TTA GAA AAG ATC TGG TCT CAA ATT GCT GAA
GAA TTT AAG        836
259    L   D   T   A   K   E   I   L   E   K   I   W   S   Q   I   A   E
E   F   K 278
837    GAT TAT GAT GAA CAC TTA ATC TTC GAA GGA TTA AAC GAA CCA AGA AAG AAT
GAT ACT CCA        896
279    D   Y   D   E   H   L   I   F   E   G   L   N   E   P   R   K   N
D   T   P 299
897    GTT GAA TGG ACT GGT GGT GAT CAA GAA GGT TGG GAT GCT GTT AAT GCT ATG
AAT GCT GTT        956
299    V    E   M   T   G   G   D   Q   E   G   W   D   A   V   N   A   M
N   A   V 318
957    TTC TTA AAG ACT GTT CGT AGT GCT GGT GGT AAT AAT CCA AAG CGT CAT CTT
ATG ATT CCA        1016
319    F   L   K   T   V   R   E   A   G   G   N   N   P   K   R   H   L
M   I   P 338
1017   CCA TAT GCT GCT GCT TGT AAT GAA AAC TCA TTC AAC AAC TTT ATC TTC CCA
GAA GAT GAT        1076
339    P    Y    A   A   A   C   N   E   N   S   F   N   N   F   I   F   P
E   D   D 358
1077   GAT AAG GTT ATT GCT TCT GTT CAT GCC TAT GCT CCA TAC AAC TTT GCC TTA
AAT AAC GGT        1136
359    D    K   V   I   A   S   V   H   A   Y   A   P   Y   N   F   A   L
N   N   G 378
1137   GAA GGA GCT GTT GAT AAG TTT GAT GCA GCT GGT AAG AGA GAT CTT GAA TGG
AAC ATT AAT        1196
379    E    G   A    V    D   K   F   D   A    A    G   K    R   D   L   E
W   N    I   N 398
1197   TTA ATG AAG AAG AGA TTT GTT GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA
TAT GGT GCT        1256
399    L   M   K   K   R   F   V   D   Q   G   I   P   M   I   L   G   E
Y   G   A 418
1257   ATG AAC CGT GAC AAT GAA GAA GAT CGT GCT ACT TGG GCT GAA TTC TAC ATG
GAA AAG GTT        1316
419    M   N   R   D   N   E   E   D   R   A   T   W   A   E   F   Y   M
E   K   V 438
1317   ACT GCT ATG GGA GTT CCA CAA ATC TGG TGG GAT AAT GGT GTC TTC GAA GGT
ACT GGT GAA        1376
439    T   A   M   G   V   P   Q   I   W   W   D   N   G   V   F   E   G
T   G   E 458
1337   CGT TTT GGT CTT CTT GAT CGT AAG AAC TTA AAG ATT GTT TAT CCA ACT ATT
GTT GCT GCT        1436
459    R    F   G   L   L   D   R   K   N   L   K   R   V   Y   P   T   I
V   A   A 478
1437   TTA CAA AAG GGT AGA GGT TTA GAA GTT AAT GTT GTT CAT GCT ATT GAA AAA
GAA ACA GAG        1496
479    L    Q    K   G   R   G   L   E   V   N   V   V   N   A   I   E   K
E   T   E 498
1497   GAA TGT TGG TCC GAA AAG TAT GGT TAT GAA TGT TGT TCA CCA AAC AAT ACT
AAG GTT GTA        155
499    E   C   W   S   E   K   Y   G   Y   E   C   C   S   P   N   N   T
K   V   V 518
1557   GTC AGT GAT GAA AGT GGT AAA TGG GGT GTT GAA AAT GGT AAC TGG TGT GGT
GTA CTC AAA        116
519    V    S   D   E   S   G   K   W   G   V   E   N   G   N   W   C   G
V   L   K 538
1617   TAC ACT GAA ACT TGT TGG TCA CTT CCA TTT GGA TAC CCA TGT TGT CCA CAT
TGT AAG GCT        1676
539    Y   T   E   T   C   W   S   L   P   F   G   Y   P   C   C   P   H
C   K   A 538
1677   CTT ACT AAG GAT GAG AAT GGT AAA TGG GGA GAA TTA AAT GGA GAA TGG TAT
GGT ATT GTT        173
559    L   T   K   D   E   N   G   K   W   G   E   L   N   G   E   W   Y
G   I   V 578
1737   GCT GAT AAA TGT TAA
attataaaataagaataaataaatttctaatgaaaaattatttaaaaaaaaataaaatag       1811
579    A   D   K   C   *          582
1812
aaaaatttatatacacatatttctaataaaatgtcatttaaaatttttatttcttattatttttaataaaaaaa
attata       1891
1892
agaaaagaaaatataaaaaataataataatgaatgaaataaaattttaattatttattcttttacttaaagcaa
aaaaaa       1971
1972
gaatttaattaaaatcaagaatttttaaagatggaatatgtattttaaataatagctaataagattataaaaat
tgtgta       2051
2052   aaaaattttaaataaaataaaaataaaataaataaataaataaataaaaaaaaaaataa
2110

This partial cDNA sequence (SEQ ID NO:1) of an eglA from Piromyces rhizinflata encodes the partial EGLA amino acid sequence (SEQ ID NO:2) shown immediately above. Analysis of the amino acid sequence encoded by the ORF indicated two nearly identical repeats, which are aligned as follows.

1      HELEWNINLMKKRFVDQGIPMILGEYGAMNRDNEEDRATWAEFYMEKVTA      50
391    RDLEWNINLMKKRFVDQGIPMILGEYGAMNRDNEEDRATWAEFYMEKVTA      440
51     MGVPQIWWDNGIFQGTGERFGLLDRKNLKIVYPTIVAALQKGRGLEVNVV      100
441    MGVPQIWWDNGVFEGTGERFGLLDRKNLKIVYPTIVAALQKGRGLEVNVV      490
101    HAVEKKPDE    109
491    HAIEKETEE    499

The two regions are amino acids 1-109 (SEQ ID NO:5) and 391-499 (SEQ ID NO:6) of EGLA. The bolded sequences in the two regions indicate identical amino acids in the alignment. It was noted that such repeats are one of the characteristics 10 of many cellulase genes (see, e.g. Aylward et al., Enzyme Microb. Technol. 24:609-614, 1999). No translation initiation codon was found at the 5' end, suggesting that the cDNA is incomplete. Using previously known cellulase genes as a model, the cDNA of pPr2301-10 clone appeared to be missing a N-terminal catalytic domain but includes a complete C-terminal catalytic domain. Based on this assumption, amino acids 110-499 of the above polypeptide sequence was considered to be a catalytic domain of EGLA and was further characterized.

The nucleic acid sequence encoding the putative EGLA catalytic domain was amplified by PCR using primers 10F (GCAGGATCCATTATGGAGCTCCCAACTAAAACTACCAAACCA; SEQ ID NO:7) and 10R (TTCCTCGAGTTAGAGCTCTTCCTCTGTTTCTTTTTCAAT; SEQ ID NO:8). To facilitate cloning, 10F contains a BamHI site, while 10R contains a XhoI site; both restriction sites are underlined in the primer sequences immediately above. The PCR product was then digested with the appropriate enzymes and ligated into BamHI and XhoI digested pGEX-4T-3 (Pharmacia Biotech, Inc.) to produce the Glutathione S-transferase (GST)-fusion expression plasmid pGEX-EGLA. The amino acid sequence downstream of the GST is shown below.

1      ATT ATG GAG CTC CCA ACT AAA ACT ACC AAA CCA ACT GAA CCA ACT GAA ACT
ACT AGT CCA        60
1      I    M   E   L   P   T   K   T   T   K   P   T   E   P   T   E   T
T   S   P 19
61     GAA GAA TCA ACT AAG CCA GAA GAA CCA ACT GGT AAT ATC CGT GAT ATT TCA
TCA AAG GAA        120
20     E   E   S   T   K   P   E   E   P    T    G    N    Z    R    D    I
S    S    K     E 39
121    TTG ATT AAG GAA ATG AAT TTC GGT TGG AAT TTA GGT AAT ACT ATG GAT GCT
CAA TGT ATT        180
40     L    I    K    E    M    N    F    G    W    N    L    G    N    T
M    D    A    Q    C    I 59
181    GAA TAC TTA AAT TAT GAA AAG GAT CAA ACT GCT TCA GAA ACT TGC TGG GGT
AAT CCA AAG        240
60     E    Y    L    N    Y    E    K    D    Q    T    A    S    E    T
C    W    G    N    P    K 79
241    ACT ACT GAA GAT ATG TTC AAG GTT TTA ATC GAC AAC CAA TTT AAT GTC TTC
CGT ATT CCA        300
80     T    T    E    D    M    F    K    V    L    I    D    N    Q    F
N    V    F    R    I    P 99
301    ACT ACT TGG TCT GGT CAC TTC GGT GAA GCT CCA GAT TAT AAG ATT GAT GAA
AAA TGG TTA        360
100    T    T    W    S    G    N    F    G    E    A    P    D    Y    K
I    D    E    K    W    L 119
361    AAG AGA GTT CAT GAA GTT GTT GAT TAT CCA TAC AAG AAC GGA GCA TTT GTT
ATC TTA AAT        420
120    K    R    V    H    E    V    V    D    Y    P    Y    K    N    G
A    F    V    I    L    N 139
421    CTT CAT CAT GAA ACC TGG AAT CAT GCC TTC TCT GAA ACT CTT GAT ACA GCC
AAG GAA ATT        480
140    L    H    H    E    T    W    N    H    A    F    S    E    T    L
D    T    A    K    E    I 159
481    TTA GAA AAG ATC TGG TCT CAA ATT GCT GAA GAA TTT AAG GAT TAT GAT GAA
CAC TTA ATC        540
160    L    E    K    I    W    E    Q    I    A    E    E    F    K    D
Y    D    E    H    L    I 179
541    TTC GAA GGA TTA AAC GAA CCA AGA AAG AAT GAT ACT CCA GTT GAA TGG ACT
GGT GGT GAT        600
180    F    E    G    L    N    E    P    R    K    N    D    T    P    V
E    W    T    G    G    D 199
601    CAA GAA GGT TGG GAT GCT GTT AAT GCT ATG AAT GCT GTT TTC TTA AAG ACT
GTT CGT AGT        660
200    Q    E    G    W    D    A    V    N    A    M    N    A    V    F
L    K    T    V    R    S 219
661    GCT GGT GGT AAT AAT CCA AAG CGT CAT CTT ATG ATT CCA CCA TAT GCT GCT
GCT TGT AAT        720
220    A    G    G    N    N    P    K    R    H    L    M    I    P    P
Y    A    A    A    C    N 239
721    GAA AAC TCA TTC AAC AAC TTT ATC TTC CCA GAA GAT GAT GAT AAG GTT ATT
GCT TCT GTT        780
240    E    N    S    F    N    N    F    I    F    P    E    D    D    D
K    V    I    A    S    V 259
761    CAT GCC TAT GCT CCA TAC AAC TTT GCC TTA AAT AAC GGT GAA GGA GCT GTT
GAT AAG TTT        840
260    H    A    Y    A    P    Y    N    F    A    L    N    N    G    E
G    A    V    D    K     F 279
841    GAT GCA GCT GGT AAG AGA GAT CTT GAA TGG AAC ATT AAT TTA ATG AAG AAG
AGA TTT GTT        900
280    D   A    A    G   K   R   D   L   E   W   N   I   N   L   M   K   K
R   F   V 299
901    GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA TAT GGT GCT ATG AAC CGT GAC
AAT GAA GAA        960
300    D   Q   G   I   P   M   I   L   G   E   Y   G   A   M   N   R   D
N   E   E 319
961    GAT CGT GCT ACT TGG GCT GAA TTC TAC ATG GAA AAG GTT ACT GCT ATG GGA
GTT CCA CAA        1020
320    D   R   A   T   W   A   E   F   Y    M   E   K   V   T   A   M   G
V   P   Q 339
1021   ATC TGG TGG GAT AAT GGT GTC TTC GAA GGT ACT GGT GAA CGT TTT GGT CTT
CTT GAT CGT        1080
340    I   W   W   D   N   G   V   F   E   G   T   G   E   R   F   G   L
L   D   R 359
1081   AAG AAC TTA AAG ATT GTT TAT CCA ACT ATT GTT GCT GCT TTA CAA AAG GGT
AGA GGT TTA        1140
360    K   N   L   K   I   V   Y    P   T   I   V   A   A   L   Q   K   G
R   G   L 379
1141   GAA GTT AAT GTT GTT CAT GCT ATT GAA AAA GAA ACA GAG GAA
1182
380    E   V   N   V   V   N   A   I   E   K   E   T   E   E 393

The complete nucleic acid sequence immediately above is designated SEQ ID NO:9, and the complete amino acid sequence encoded by that nucleic acid sequence is designated SEQ ID NO:10. Nucleotides 13-1182 of SEQ ID NO:9 (SEQ ID NO:3) correspond to nucleotides 331-1499 of SEQ ID NO:1. Amino acids 5-393 of SEQ ID NO:10 (SEQ ID NO:4) correspond to amino acids 110-499 of SEQ ID NO2.

The EGLA catalytic domain expression plasmid was used to transformed E. coli to produce recombinant EGLA. GST-EGLA was purified on glutathione Sepharose 4B (Pharmacia Biotech, Inc.) following the manufacturer's protocols. Bound fusion protein was cleaved with thrombin to release only the EGLA catalytic domain.

The enzymatic activity of the EGLA fragment was determined as follows. The purified protein was suspended in 50 mM sodium phosphate buffer containing 1% CMC, 1% oat spelt xylan, 0.4% barley β-glucan, 1% lechinan, 5 mM pNP-β-D-glucoside, Avicel, or 5 mM pNP-β-D-cellobioside. The barley β-glucan contains mixed β-1,3'-1,4' glucan. Enzymatic activity was measured by detecting the amount of reducing sugar released from the substrate. After incubating the reaction at 50°C for 10 minutes, the reaction was stopped by adding a half-volume each of 0.3% (w/v) 3,6-dinitrophthalic acid and stop solution (25% K₂ CO₃ and 5% Na₂ S₂ O₃). The stopped reaction was then boiled for 10 minutes before absorbance at 450 nm was read. Protein concentrations were measured using a protein assay kit (BioRad). The results are summarized in Table 1 below.

TABLE 1
Specific Activity    Relative Activity
Substrate          (μmoles glucose/mg/min) (%)
Carboxymethyl      590.8                100
cellulose
Barley β-glucan 745.7                126.2
Lechinan           565.7                95.8
Oat Spelt Xylan    127.6                21.6

No activity was detected using pNP-β-D-glucoside, Avicel, or pNP-β-D-cellobioside as a substrate under these conditions.

Using the assay described immediately above, the temperature or pH was varied to obtain conditions necessary for optimal activity. The optimal temperature for the EGLA catalytic domain was about 50°C, and the optimal pH for the catalytic domain was about 5.5. In addition, EGLA retained about 30% activity against the substrate carboxymethyl cellulose and 42% activity against the substrate barley β-glucan after the enzyme was heated to 80°C for 10 min, indicating that the EGLA catalytic domain described here is moderately heat stable.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of this invention.

Claims

1. An isolated nucleic acid encoding a polypeptide, the amino acid sequence of which is at least 95% identical to SEQ ID NO:4, wherein said polypeptide hydrolyzes a polysaccharide containing a β-1,3' or β-1,4' glycosidic linkage.

2. The isolated nucleic acid of claim 1 encoding a polypeptide, the amino acid sequence of which is SEQ ID NO:4.

3. The isolated nucleic acid of claim 2, wherein the nucleic acid sequence is SEQ ID NO:3.